This invention relates to chemical and biochemical assays involving one or more ligands that will specifically bind to respective reactive moieties of interest.
A number of known chemical or biochemical analyte/ligand reactions in which a complex is formed by the reaction of moieties that will highly specifically bind to one another, are used as the basis for biochemical or chemical assays. Particularly, the well known immune reaction involving the formation of antibody-antigen complexes, nucleic acid hybridizations, enzyme-inhibitor, enzyme-coenzyme, hormone-receptor, enzyme-receptor and like substrate-specific reactions are exemplary of some of those known specific binding reactions. The terms "moiety" and "ligand" as used herein generally interchangeably refer to the reactive portions of such complex; with respect to an antigen-antibody complex, for example, these terms are intended to include all immunologically reactive portions such as haptens, complete antigens, reactive antigen-antibody fragments, and complete antibodies. The term "analyte" is intended to refer to the moiety or ligand being assayed either qualitatively, quantitatively or both, and the term "captor" is intended to refer to the ligand or moiety that will specifically bind to the analyte of interest.
Assays based upon these well known specific binding reactions involve a wide variety of techniques. Some assay methods employ radioactive, luminescent or fluorescent tags that are coupled to either the ligand or to the analyte. Those tags can be detected or measured from the radiation arising from the reaction product or complex. For example, a fluorescence assay system using an attenuated total reflection (ATR) cell is shown and described in detail in U.S. Pat. No. 4,558,014 issued Dec. 10, 1985 and is incorporated herein by reference.
Where such a typical assay cell is a disposable unit used for a single sample assay, in a number of instances the captor ligands may not be covalently bonded to the substrate, but may simply be adsorbed thereon for convenience, cost or other reasons. Contacting the cell with the sample containing the analyte, however, may wash off some of the adsorbed captors, reducing the signal so that it becomes difficult to correlate signal strength with concentration from sample to sample.
Further, stringent quality control measures are usually required in order to manufacture single-assay, disposable, coated cells that respond substantially identically. In practice, one samples a large population of such cells, and then checks the cells with a standardized assay to see if the sampled cells respond within a desired tolerance. It also may not be either necessary or desirable to manufacture cells that are disposable after each use, and in such case consideration should be given to sequential use of a cell to assay several different samples.
Accordingly, a principal object of the present invention is to provide a method of readily and easily determining the amount or concentration of the immobilized ligand on the substrate for use in a capture assay. Another object of the present invention is to provide an assay method that will permit the determination of both the concentration of the immobilized ligand as well as providing a measurement of the analyte in a sample, which analyte will specifically bind with such ligand.
Other objects of the present invention will in part be obvious and will in part appear hereinafter.